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Actual. osteol ; 19(2): 128-143, sept. 2023. ilus, tab
Article in Spanish | LILACS, UNISALUD, BINACIS | ID: biblio-1523882


El presente trabajo muestra la obtención de un material a partir de un polímero sintético (TerP) y otro natural, mediante entrecruzamiento físico y su caracterización fisicoquímica y biológica, con el fin de emplearlos para regeneración de tejido óseo. Las membranas fueron obtenidas por la técnica de evaporación del solvente y caracterizadas por espectroscopia FTIR, ensayos de hinchamiento, medidas de ángulo de contacto y microscopia electrónica de barrido (SEM). Se encontró que la compatibilidad entre los polímeros que la constituyen es estable a pH fisiológico y que, al incorporar mayor cantidad del TerP a la matriz, esta se vuelve más hidrofóbica y porosa. Además, teniendo en cuenta la aplicación prevista para dichos materiales, se realizaron estudios de biocompatibilidad y citotoxicidad con células progenitoras de médula ósea (CPMO) y células RAW264.7, respectivamente. Se evaluó la proliferación celular, la producción y liberación de óxido nítrico (NO) al medio de cultivo durante 24 y 48 horas y la expresión de citoquinas proinflamatorias IL-1ß y TNF-α de las células crecidas sobre los biomateriales variando la cantidad del polímero sintético. Se encontró mayor proliferación celular y menor producción de NO sobre las matrices que contienen menos proporción del TerP, además de poseer una mejor biocompatibilidad. Los resultados de este estudio muestran que el terpolímero obtenido y su combinación con un polímero natural es una estrategia muy interesante para obtener un biomaterial con posibles aplicaciones en medicina regenerativa y que podría extenderse a otros sistemas estructuralmente relacionados. (AU)

In the present work, the preparation of a biomaterial from a synthetic terpolymer (TerP) and a natural polymer, physically crosslinked, is shown. In order to evaluate the new material for bone tissue regeneration, physicochemical and biological characterizations were performed. The membranes were obtained by solvent casting and characterized using FTIR spectroscopy, swelling tests, contact angle measurements, and scanning electron microscopy (SEM). It was found that the compatibility between the polymers is stable at physiological pH and the incorporation of a higher amount of TerP into the matrix increases hydrophobicity and porosity.Furthermore, considering the intended application of these materials, studies of biocompatibility and cytotoxicity were conducted with Bone Marrow Progenitor Cells (BMPCs) and RAW264.7 cells, respectively. Cell proliferation, NO production and release into the culture medium for 24 and 48 hours, and proinflammatory cytokine expression of IL-1ß and TNF-α from cells grown on the biomaterials while varying the amount of the synthetic polymer were evaluated. Greater cell proliferation and lower NO production were found on matrices containing a lower proportion of TerP, in addition to better biocompatibility. The results of this study demonstrate that the obtained terpolymer and its combination with a natural polymer is a highly interesting strategy for biomaterial preparation with potential applications in regenerative medicine. This approach could be extended to other structurally related systems. (AU)

Animals , Rats , Osteogenesis , Polymers/chemistry , Biocompatible Materials/chemical synthesis , Bone and Bones/chemistry , Bone Regeneration , Chitosan/chemistry , Polymers/toxicity , Biocompatible Materials/toxicity , Materials Testing , Cell Differentiation , Chromatography, Gel , Spectroscopy, Fourier Transform Infrared , Cell Culture Techniques , Nuclear Magnetic Resonance, Biomolecular , Chitosan/toxicity
Rev. Ciênc. Saúde ; 13(3): 23-30, 20230921.
Article in English | LILACS | ID: biblio-1510835


Objective: The present study aims to evaluate the viability of adult human neural cells in culture obtained from traumatized brain tissues collected in emergency surgery procedures. Methods: Exploratory, descriptive, quantitative and cross-sectional study evaluating samples obtained from patients who underwent traumatic brain injury with extrusion of brain tissue submitted to cell culture in a standardized medium, being preserved during 168h. After observation under phase contrast microscopy and immunohistochemical processing for neuronal (MAP-2) and glial (GFAP) markers, morphometric parameters of neural cells (cell body area, dendritic field length and fractal dimension) were evaluated using ImageJ software, with data obtained after 24, 72 and 168h being compared using non-parametric Kruskal Wallis test, followed by Dunn's post hoc test. Results: The explant of the nervous tissue revealed a consolidated pattern of cell migration into the culture medium. Cell proliferation, upon reaching confluence, presented an aspect of cellular distribution juxtaposed along the culture medium at all time points analyzed. Both neurons and glial cells remained viable after 168h in culture, with their morphologies not varying significantly throughout the time points evaluated. Immunohistochemistry for MAP-2 showed a relatively well-preserved cytoskeletal organization. GFAP immunoreactivity revealed activated astrocytes especially at the later time point. Conclusions: Our results point out the viability of cell culture from traumatized human nervous tissue, opening up perspectives for the use of substances of natural origin that may contribute neuroprotectively to neuronal maintenance in culture, allowing future translational approach.

Humans , Male , Adult , Brain Injuries , Cell Culture Techniques , Neurons , Wounds and Injuries , Traumatology , Immunohistochemistry
Chinese Journal of Oncology ; (12): 464-470, 2023.
Article in Chinese | WPRIM | ID: wpr-984745


Conventional tumor culture models include two-dimensional tumor cell cultures and xenograft models. The former has disadvantages including lack of tumor heterogeneity and poor clinical relevance, while the latter are limited by the slow growth, low engraftment successful rate, and high cost. In recent years, in vitro three-dimensional (3D) tumor models have emerged as the tool to better recapitulate the spatial structure and the in vivo environment of tumors. In addition, they preserve the pathological and genetic features of tumor cells and reflect the complex intracellular and extracellular interactions of tumors, which have become a powerful tool for investigating the tumor mechanism, drug screening, and personalized cancer treatment. 3D tumor model technologies such as spheroids, organoids, and microfluidic devices are maturing. Application of new technologies such as co-culture, 3D bioprinting, and air-liquid interface has further improved the clinical relevance of the models. Some models recapitulate the tumor microenvironment, and some can even reconstitute endogenous immune components and microvasculature. In recent years, some scholars have combined xenograft models with organoid technology to develop matched in vivo/in vitro model biobanks, giving full play to the advantages of the two technologies, and providing an ideal research platform for individualized precision therapy for specific molecular targets in certain subtypes of tumors. So far, the above technologies have been widely applied in the field of colorectal cancer research. Our research team is currently studying upon the application of patient-derived tumor cell-like clusters, a self-assembly 3D tumor model, in guiding the selection of postoperative chemotherapy regimens for colorectal cancer. A high modeling success rate and satisfactory results in the drug screening experiments have been achieved. There is no doubt that with the advancement of related technologies, 3D tumor models will play an increasingly important role in the research and clinical practice of colorectal cancer.

Humans , Organoids/pathology , Cell Culture Techniques , Colorectal Neoplasms/pathology , Tumor Microenvironment
Chinese Journal of Hepatology ; (12): 668-672, 2023.
Article in Chinese | WPRIM | ID: wpr-986190


Liver fibrosis incidence and adverse outcomes are high; however, there are no known chemical drugs or biological agents that are specific and effective for treatment. The paucity of a robust and realistic in vitro model for liver fibrosis is one of the major causes hindering anti-liver fibrosis drug development. This article summarizes the latest progress in the development of in vitro cell models for liver fibrosis, with a focus based on the analysis of induction and activation of hepatic stellate cells, cell co-culture, and 3D model co-construction, as well as concurrent potential methods based on hepatic sinusoidal endothelial cell establishment.

Humans , Liver Cirrhosis/pathology , Hepatic Stellate Cells , Cell Culture Techniques , Endothelial Cells
Journal of Biomedical Engineering ; (6): 602-608, 2023.
Article in Chinese | WPRIM | ID: wpr-981582


Three-dimensional (3D) cell culture model is a system that co-culture carriers with 3D structural materials and different types of cells in vitro to simulate the microenvironment in vivo. This novel cell culture model has been proved to be close to the natural system in vivo. In the process of cell attachment, migration, mitosis and apoptosis, it could produce biological reactions different from that of monolayer cell culture. Therefore, it can be used as an ideal model to evaluate the dynamic pharmacological effects of active substances and the metastasis process of cancer cells. This paper compared and analyzed the different characteristics of cell growth and development under two-dimensional (2D) and 3D model culture and introduced the establishment method of 3D cell model. The application progress of 3D cell culture technology in tumor model and intestinal absorption model was summarized. Finally, the application prospect of 3D cell model in the evaluation and screening of active substance was revealed. This review is expected to provide reference for the development and application of new 3D cell culture models.

Cell Culture Techniques, Three Dimensional , Cell Culture Techniques , Apoptosis , Cell Proliferation , Technology
Hematol., Transfus. Cell Ther. (Impr.) ; 44(4): 560-566, Oct.-dec. 2022. tab, graf
Article in English | LILACS | ID: biblio-1421532


ABSTRACT Introduction: Culturing bone marrow mesenchymal stem cells (BM-MSCs) is a key point in different fields of research, including tissue engineering and regenerative medicine and studies of the bone marrow microenvironment. However, isolating and expanding murine BM-MSCs in vitro has challenged researchers due to the low purity and yield of obtained cells. In this study, we aimed to evaluate five different protocols to culture murine BM-MSCs in vitro. Methods: All protocols were based on the adhesion capacity of BM-MSCs to the tissue culture plastic surface and varied in the types of plate, culture media, serum, additional supplementation and initial cell density. Flow cytometry analysis was used to investigate lineage purity after expansion. Results: The expression of CD45 and CD11b was detected in the cultures generated according to all protocols, indicating low purity with the presence of hematopoietic cells and macrophages. The cellular growth rate and morphology varied between the cultures performed according to each protocol. Cells cultured according to protocol 5 (8 × 107cells/plate, Roswell Park Memorial Institute (RPMI) culture medium during first passage and then Iscove's Modified Delbecco's Medium (IMDM) culture medium, both supplemented with 9% fetal bovine serum, 9% horse serum, 12μM L-glutamine) presented the best performance, with a satisfactory growth rate and spindle-shape morphology. Conclusion: Our results point out that the purity and satisfactory growth rate of murine BM-MSC cultures are not easily achieved and additional approaches must be tested for a proper cell expansion.

Animals , Male , Rats , Mesenchymal Stem Cells , Bone Marrow , In Vitro Techniques , Cell Culture Techniques , Mice
São Paulo; s.n; 20220601. 109 p.
Thesis in Portuguese | LILACS, BBO | ID: biblio-1370762


O carcinoma secretório em glândula salivar é uma neoplasia recentemente descrita que tem os mesmos aspectos morfológicos, imuno-histoquímicos e genéticos do carcinoma secretório de origem mamária. O carcinoma secretório tem características celulares reminiscentes de uma célula secretora lactacional, isto é, um citoplasma vacuolado repleto de gotas lipídicas e um material secretado, por vezes de forma apócrina, que pode lembrar o leite. Mais recentemente, algum nível de diferenciação lactacional foi sugerida no carcinoma secretório de origem salivar. O objetivo do estudo foi verificar se existe uma diferenciação do tipo lactacional em carcinomas secretórios de origem salivar, comparando a outros tipos de carcinomas salivares mais comuns. Foram realizadas reações imuno-histoquímicas para as seguintes proteínas: receptores hormonais (receptor de prolactina e receptor do hormônio do crescimento), proteínas associadas ao produto de secreção da glândula mamária lactacional (mucina-1 (MUC-1), MUC4, globulina de gordura 1 do leite humano, lactoferrina) e proteínas associadas à via Akt-mTOR (PTEN, p-Akt, p-mTOR, p4EBP1, eIF4E, pS6). A maioria dos casos de carcinoma secretório foi negativa para receptor de prolactina e de hormônio do crescimento. Lactoferrina foi positiva em todos os grupos tumorais, porém somente em carcinoma secretório observou-se um padrão de marcação intensa, difuso tanto em célula como em secreção. Todos os casos de carcinoma secretório foram positivos para globulina de gordura do tipo 1, porém o mesmo padrão de marcação foi observado em outros tumores. A maioria dos casos de carcinoma secretório foram positivos para MUC1 e MUC4. Nenhum caso de carcinoma secretório foi positivo para Akt, mas PTEN foi difusamente expresso em 57,1% dos casos. mTOR foi expresso em mais da metade dos casos de carcinoma secretório e dos outros tumores salivares. Entre as proteínas à jusante de mTOR, somente eIF4E demonstrou alta expressão no grupo de estudo. A expressão de marcadores lactacionais não é exclusiva do carcinoma secretório, porém a expressão de lactoferrina é distinta neste grupo de tumores quando comparado aos demais tumores salivares estudados.

Cell Culture Techniques , Diphosphonates , Glucocorticoids
Int. j. morphol ; 40(1): 91-97, feb. 2022. ilus
Article in English | LILACS | ID: biblio-1385597


SUMMARY: Carnosine is known as a natural dipeptide, which inhibits the proliferation of tumor cells throughout its action on mitochondrial respiration and cell glycolysis. However, not much is known about its effects on the metabolism of healthy cells. We explored the effects of Karnozin EXTRA® capsule with different concentrations of L-carnosine, on the cell viability and the expressions of intermediate filament vimentin (VIM) and superoxide dismutase (SOD2) in normal fibroblasts BHK-21/C13. Furthermore, we investigated its action on the energy production of these cells. Cell viability was quantified by the MTT assay. The Clark oxygen electrode (Oxygraph, Hansatech Instruments, England) was used to measure the "intact cell respiration rate", state 3 of ADP-stimulated oxidation, maximum oxidation capacity and the activities of complexes I, II and IV. Results showed that Karnozin EXTRA® capsule in concentrations of 2 and 5 mM of L-carnosine did not induce toxic effects and morphological changes in treated cells. Our data revealed a dose-dependent immunofluorescent signal amplification of VIM and SOD2 in the BHK-21/C13 cell line. This supplement substantially increased the recorded mitochondrial respiration rates in the examined cell line. Due to the stimulation of mitochondrial energy production in normal fibroblasts, our results suggested that Karnozin EXTRA® is a potentially protective dietary supplement in the prevention of diseases with altered mitochondrial function.

RESUMEN: La carnosina se conoce como dipéptido natural, que inhibe la proliferación de células tumorales a través de su acción sobre la respiración mitocondrial y la glucólisis celular. Sin embargo, no se sabe mucho de sus efectos sobre el metabolismo de las células sanas. Exploramos los efectos de la cápsula Karnozin EXTRA® con diferentes concentraciones de L-carnosina, sobre la viabilidad celular y las expresiones de vimentina de filamento intermedio (VIM) y superóxido dismutasa (SOD2) en fibroblastos normales BHK-21 / C13. Además, estudiamos su acción sobre la producción de energía de estas células. La viabilidad celular se cuantificó mediante el ensayo MTT. Se utilizó el electrodo de oxígeno Clark (Oxygraph, Hansatech Instruments, Inglaterra) para medir la "tasa de respiración de células intactas", el estado 3 de oxidación estimulada por ADP, la capacidad máxima de oxidación y las actividades de los complejos I, II y IV. Los resultados mostraron que la cápsula de Karnozin EXTRA® en concentraciones de 2 y 5 mM de L- carnosina no indujo efectos tóxicos ni cambios morfológicos en las células tratadas. Nuestros datos revelaron una amplificación de señal inmunofluorescente dependiente de la dosis de VIM y SOD2 en la línea celular BHK-21 / C13. Este suplemento aumentó sustancialmente las tasas de respiración mitocondrial registradas en la línea celular examinada. Debido a la estimulación de la producción de energía mitocondrial en fibroblastos normales, nuestros resultados sugirieron que Karnozin EXTRA® es un suplemento dietético potencialmente protector en la prevención de enfermedades con función mitocondrial alterada.

Animals , Carnosine/pharmacology , Fibroblasts/drug effects , Kidney/cytology , Superoxide Dismutase/drug effects , Vimentin/drug effects , Biological Assay , Cell Survival/drug effects , Fluorescent Antibody Technique , Cricetinae , Cell Culture Techniques , Energy Metabolism
Bol. latinoam. Caribe plantas med. aromát ; 21(1): 108-122, ene. 2022. ilus, tab
Article in English | LILACS | ID: biblio-1372494


Cota tinctoria is a medicinal plant which has been used for management of cancer in folk medicine of various regions. The aim of present study is to investigate cytotoxic activity of different concentrations of hydroalcoholic extract of C. tinctoria flowers on gastric (AGS) and liver (Hep-G2) cancer cell lines as well as Human Natural GUM fibroblast (HUGU) cells. Cell mortality rates were examined after 24, 48 and 72 h incubations using the MTT assay. IC50of extract on AGS cells after 24, 48 and 72h was 1.46, 1.29 and 1.14 µg/mL respectively. The extract demonstrated IC50 of 5.15, 3.92 and 2.89 µg/mL on Hep-G2 cells after 24, 48 and 72 h respectively. No cytotoxic effect was detected on HUGU (Human Natural GUM fibroblast) cells. C. tinctoria seems to have a promising potential to be considered as a source for anticancer drug discovery. However, more experimental and clinical studies are required.

Cota tinctoria es una planta medicinal que se ha utilizado para el tratamiento del cáncer en la medicina popular de varias regiones. El objetivo del presente estudio es investigar la actividad citotóxica de diferentes concentraciones de extracto hidroalcohólico de flores de C. tinctoria en líneas celulares de cáncer gástrico (AGS) e hígado (Hep-G2), así como en células de fibroblasto GUM humano natural (HUGU). Se examinaron las tasas de mortalidad celular después de incubaciones de 24, 48 y 72 h utilizando el ensayo MTT. La CI50 del extracto en células AGS después de 24, 48 y 72 h fue de 1,46; 1,29 y 1,14 µg respectivamente. El extracto demostró una CI50 de 5,15, 3,92 y 2,89 µg/mL en células Hep-G2 después de 24, 48 y 72 h, respectivamente. No se detectó ningún efecto citotóxico en las células HUGU (fibroblasto GUM humano natural). C. tinctoria parece tener un potencial prometedor para ser considerada como una fuente de descubrimiento de fármacos contra el cáncer. Sin embargo, se requieren más estudios experimentales y clínicos.

Plant Extracts/administration & dosage , Asteraceae/chemistry , Cell Line, Tumor/drug effects , Liver Neoplasms/drug therapy , Antineoplastic Agents, Phytogenic/administration & dosage , Stomach Neoplasms/drug therapy , Flavonoids/analysis , Plant Extracts/pharmacology , Plant Extracts/chemistry , Cell Culture Techniques , Anthemis/chemistry , Phenolic Compounds/analysis , Hep G2 Cells/drug effects , Antineoplastic Agents, Phytogenic/pharmacology , Antineoplastic Agents, Phytogenic/chemistry
Chinese Journal of Stomatology ; (12): 535-539, 2022.
Article in Chinese | WPRIM | ID: wpr-935898


Salivary glands are important organs in the oral and maxillofacial region. Environment and genetic factors may cause salivary gland tumors or non-neoplastic diseases, but the mechanisms of those diseases are still unclear. One of the important reasons is the short of researching media and model. As a new technique and research model, organoids have been widely used in the research of various diseases. Organoid culture plays a bridging role between two-dimensional cell culture and living animal models, and it is also the most promising translational research model that could connect the clinical research to basic research. This review will discuss the recent development of organoid techniques in the culture of normal salivary glands and salivary gland tumors, also their applications and challenges in tissue engineering, etiological research, and tumor therapy.

Animals , Cell Culture Techniques , Organoids , Salivary Gland Neoplasms , Salivary Glands , Tissue Engineering
Chinese Journal of Traumatology ; (6): 138-144, 2022.
Article in English | WPRIM | ID: wpr-928493


PURPOSE@#The incidence of acute lung injury (ALI) in severe trauma patients is 48% and the mortality rate following acute respiratory distress syndrome evolved from ALI is up to 68.5%. Alveolar epithelial type 1 cells (AEC1s) and type 2 cells (AEC2s) are the key cells in the repair of injured lungs as well as fetal lung development. Therefore, the purification and culture of AEC1s and AEC2s play an important role in the research of repair and regeneration of lung tissue.@*METHODS@#Sprague-Dawley rats (3-4 weeks, 120-150 g) were purchased for experiment. Dispase and DNase I were jointly used to digest lung tissue to obtain a single-cell suspension of whole lung cells, and then magnetic bead cell sorting was performed to isolate T1α positive cells as AEC1s from the single-cell suspension by using polyclonal rabbit anti-T1a (a specific AEC1s membrane protein) antibodies combined with anti-rabbit IgG microbeads. Afterwards, alveolar epithelial cell membrane marker protein EpCAM was designed as a key label to sort AEC2s from the remaining T1α-neg cells by another positive immunomagnetic selection using monoclonal mouse anti-EpCAM antibodies and anti-mouse IgG microbeads. Cell purity was identified by immunofluorescence staining and flow cytometry.@*RESULTS@#The purity of AEC1s and AEC2s was 88.3% ± 3.8% and 92.6% ± 2.7%, respectively. The cell growth was observed as follows: AEC1s stretched within the 12-16 h, but the cells proliferated slowly; while AEC2s began to stretch after 24 h and proliferated rapidly from the 2nd day and began to differentiate after 3 days.@*CONCLUSION@#AEC1s and AEC2s sorted by this method have high purity and good viability. Therefore, our method provides a new approach for the isolation and culture of AEC1s and AEC2s as well as a new strategy for the research of lung repair and regeneration.

Animals , Rats , Alveolar Epithelial Cells/cytology , Cell Culture Techniques , Cell Separation/methods , Immunoglobulin G/metabolism , Lung , Magnetic Phenomena , Rats, Sprague-Dawley
Chinese Journal of Preventive Medicine ; (12): 20-24, 2022.
Article in Chinese | WPRIM | ID: wpr-935244


In the process of xenobiotic toxicity prediction and risk assessment, in vitro cell culture models possess high practical application value. With the rapid development of biological technologies such as three-dimensional (3D) bio-printing, organoid culture and organ-on-a-chip systems, in vitro cell culture models have made great progress. Sharing the similarities in structure, function and the physiological environment with tissues or organs in vivo, hazard identification and dose-response analysis based on 3D cell culture models provide access to more accurate toxicity data as a theoretical basis for risk assessment and risk management of chemicals. This review summarizes the establishment of three typical 3D cell culture models, i.e., human cell line-based co-culture model, 3D-printed scaffold-based cell culture model and organoids, and their application in toxicity tests of xenobiotics.

Humans , Cell Culture Techniques , Cell Culture Techniques, Three Dimensional , Cell Line , Toxicity Tests , Xenobiotics/toxicity
Braz. J. Pharm. Sci. (Online) ; 58: e19660, 2022. tab, graf
Article in English | LILACS | ID: biblio-1394027


Abstract In an attempt to increase molecular stability and provide controlled release, vascular endothelial growth factor (VEGF) was encapsulated into polycaprolactone (PCL) nanoparticles. Both VEGF-free and VEGF-loaded PCL nanoparticles were formulated by w/o/w double emulsion of the dichloromethane-water system in the presence of polyvinyl alcohol (PVA) and rat serum albumin. To achieve the optimal formulation concerning particle size and monodispersity, studies were carried out with different formulation parameters, including PVA concentration, homogenization time and rate. Scanning electron microscopy and dynamic light scattering analysis showed respectively that particles had a spherical shape with a smooth surface and particle size varying between 58.68-751.9 nm. All of the formulations were negatively charged according to zeta potential analysis. In vitro release study was performed in pH 7.4 phosphate-buffered saline at 37°C and released VEGF amount was measured by enzyme-linked immunosorbent assay (ELISA) method. At the end of the 35th day, 10% of total encapsulated VEGF was released with a sustained-release profile, which fitted the Korsmeyer-Peppas kinetic model. The bioactivation of the nanoparticles was evaluated using XTT and ELISA methods. As a result, the released VEGF was biologically active and also VEGF loaded PCL nanoparticles enhanced proliferation of the human umbilical vein endothelial cells in cell culture.

Vascular Endothelial Growth Factor A , Nanoparticles/classification , In Vitro Techniques/methods , Enzyme-Linked Immunosorbent Assay/methods , Microscopy, Electron, Scanning/methods , Cell Culture Techniques/methods , Human Umbilical Vein Endothelial Cells
Braz. J. Pharm. Sci. (Online) ; 58: e191120, 2022. tab, graf
Article in English | LILACS | ID: biblio-1394048


Abstract The aim of the current study was to assess the physicochemical characteristics and wound healing activity of chitosan-polyvinyl alcohol (PVA) crosslinked hydrogel containing recombinant human epidermal growth factor (rh-EGF) or recombinant mouse epidermal growth factor (rm-EGF). The hydrogels were prepared and analyses were made of the morphological properties, viscosity, water absorption capacity, mechanical and bio-adhesive properties. The viscosity of the formulations varied between 14.400 - 48.500 cPs, with the greatest viscosity values determined in K2 formulation. F2 formulation showed the highest water absorption capacity. According to the studies of the mechanical properties, H2 formulation (0.153±0.018 showed the greatest adhesiveness and E2 (0.245±0.001 mj/cm2) formulation, the highest bio-adhesion values. Hydrogels were cytocompatible considering in vitro cell viability values of over 76% on human keratinocyte cells (HaCaT, CVCL-0038) and of over 84% on human fibroblast cells (NIH 3T3, CRL-1658) used as a model cell line. According to the BrdU cell proliferation results, B1 (197.82±2.48%) formulation showed the greatest NIH 3T3 and C1 (167.43±5.89%) formulation exhibited the highest HaCaT cell proliferation ability. In addition, the scratch closure assay was performed to assess the wound healing efficiency of formulation and the results obtained in the study showed that F2 formulation including PEGylated rh-EGF had a highly effective role.

Wound Healing , Hydrogels/analysis , Chitosan/chemical synthesis , Epidermal Growth Factor , Polyvinyl Alcohol/pharmacology , Wounds and Injuries/classification , In Vitro Techniques/methods , Cell Culture Techniques/methods , Cell Proliferation/genetics , Absorption
São Paulo; s.n; s.n; 2022. 56 p. tab, graf.
Thesis in Portuguese | LILACS | ID: biblio-1396952


O câncer de pele pode ser classificado como não melanoma e melanoma. O melanoma apresenta baixa incidência entre os cânceres de pele, porém é a forma mais letal e é considerado um dos tipos mais resistentes ao tratamento. Devido à infiltração de células malignas nos tecidos, vasos linfáticos e vasos sanguíneos, o melanoma invade e se espalha rapidamente. Suas metástases são frequentemente localizadas em linfonodos, cérebro, fígado e outros órgãos. Melanomas metastáticos abrigam múltiplas mutações gênicas e muitos tumores apresentam resistência aos tratamentos, como por exemplo com inibidores BRAF, devido à mutações e ativação de vias paralelas. Ou seja, existe uma necessidade clara da busca de novas opções de tratamento. Em trabalho realizado por nosso grupo, Massaro et al mostraram que o derivado de estradiol 2- Metoxiestradiol induz apoptose em células de melanoma e senescência. Neste sentido, o composto STX140, (um análogo do estradiol com biodisponibilidade superior), que já se mostrou eficaz no combate ao câncer de mama em diversos estudos in vitro e in vivo, será então avaliado para sua ação no melanoma de forma inédita. Este trabalho teve como principal objetivo explorar a ação antitumoral em células de melanoma do composto STX140, especialmente a indução de senescência. Utilizando a cultura de células de melanoma foram realizados os ensaios de: viabilidade celular - IC50, formação de colônias, análise do ciclo celular e caracterização de morte celular por citometria de fluxo, ensaio In vitro scratch, coloração para ß-galactosidase, PCR quantitativo e ELISA. Os resultados mostraram que o composto STX140: diminui a viabilidade celular, inibe a proliferação, formação de colônias e migração em linhagens de melanoma (não resistentes e resistentes ao vemurafenibe, inibidor de BRAF). Além do mais, o composto atuou diminuindo a secreção da interleucina pró-tumoral IL-8 em células resistentes. O STX140 induziu senescência nas células de melanoma que foram positivas para ß-galactosidase, também havendo aumento da expressão de genes chave de vias de senescência (CDKN1A e GADD45A) nas células de melanoma resistentes tratadas com o composto. Em conclusão, o STX140 mostrou ter um potencial antitumoral contra o melanoma, diminuindo sua viabilidade celular, inibindo sua proliferação e migração, induzindo senescência, diminuindo a secreção de interleucina pró- tumoral, com efeito mais acentuado nas linhagens de melanoma resistente

Skin cancer can be classified as non-melanoma and melanoma. Melanoma has a low incidence among skin cancers, but it is the most lethal form and is considered one of the most resistant to treatment. Due to the infiltration of malignant cells into tissues, lymphatic vessels and blood vessels, melanoma invades and spreads rapidly. Its metastases are often located in lymph nodes, brain, liver and other organs. Metastatic melanomas presents multiple gene mutations and many tumors are resistant to treatments, such as with BRAF inhibitors, due to mutations and activation of parallel pathways. In other words, there is a clear need to search for new treatment options. In work carried out by our group, Massaro et al showed that the estradiol derivative 2- Methoxyestradiol induces apoptosis in melanoma cells and senescence. In this sense, the compound STX140, (an estradiol analogue with superior bioavailability), which has already been shown to be effective against breast cancer in vitro and in vivo studies will be then evaluated for its action on melanoma. The main objective of this work is to explore the antitumor action of the compound STX140 in melanoma cells, especially the induction of senescence. Using the melanoma cell culture the following assays were performed: cell viability - IC50, clonogenic, cell cycle analysis and cell death characterization by flow cytometry, wound assay, staining for ß-galactosidase, quantitative PCR and ELISA. Preliminary data from this work showed that the compound STX140: decreases cell viability, inhibits proliferation, colony formation and migration in melanoma cell lines (non-resistant and resistant to vemurafenib, BRAF inhibitor). It also decreased the secretion of pro-tumor interleukin IL-8 in resistant cells. STX140 induced senescence in melanoma cells, that were positive for ß-galactosidase, and there was also increased expression of key genes of senescence pathways (CDKN1A and GADD45A) in resistant melanoma cells treated with the compound. In conclusion, STX140 has been shown to have antitumor potential against melanoma, decreasing its cell viability, inhibiting its proliferation and migration, inducing senescence, decreasing pro-tumor interleukin secretion, with a more pronounced effect on resistant melanoma cell lines

Estradiol/analogs & derivatives , Melanoma/pathology , Skin Neoplasms/pathology , In Vitro Techniques/methods , Aging/metabolism , Interleukin-8/adverse effects , Cell Culture Techniques/methods , Inhibitory Concentration 50 , Flow Cytometry/instrumentation , Neoplasm Metastasis
Bol. latinoam. Caribe plantas med. aromát ; 20(5): 515-523, sept. 2021. ilus
Article in English | LILACS | ID: biblio-1369061


To explore a new underlying molecular mechanism of Huangkui Extract Powder (HKEP) in the alleviation of diabetic nephropathy (DN). Murine immortalized podocytes were divided into (i) normal glucose (NG, 5.6 mM), (ii) NG + HKEP (0.45 g/L), (iii) HG, and (iv) HG + HKEP (0.45 g/L) groups. MTT assay and flow cytometry were used to detect the podocyte proliferation, apoptosis and cell cycle. Cell viability was inhibited, and apoptosis increased in(iii) HG group compared with (i) NG group (p<0.05). mRNA and protein expression of nephrin and podocin significantly decreased in (iii) HG group compared with (i) NG group (p<0.05). When compared with (iii) HG group, (iv) HG + HKEP group had higher cell viability, lower apoptotic rate and higher mRNA and protein expression of nephrin and podocin (p<0.05). HKEP can attenuate HG-induced podocyte damage, which may be one of the mechanisms of HKEP for attenuating DN.

Explorar un nuevo mecanismo molecular subyacente del extracto del polvo de Huangkui (HKEP) en el alivio de la nefropatía diabética (ND). Los podocitos murinos inmortalizados se dividieron en (i) grupos de glucosa normal (NG, 5,6 mM), (ii) NG + HKEP (0,45 g/L), (iii) HG y (iv) HG + HKEP (0,45 g/L). Se utilizaron el ensayo MTT y la citometría de flujo para detectar la proliferación de podocitos, la apoptosis y el ciclo celular. La viabilidad celular se inhibió y la apoptosis aumentó en el grupo (iii) HG en comparación con el grupo (i) NG (p<0,05). La expresión de ARNm y proteínas de nefrina y podocina disminuyó significativamente en el grupo (iii) HG en comparación con el grupo (i) NG (p<0,05). En comparación con el grupo (iii) HG, el grupo (iv) HG + HKEP tuvo una mayor viabilidad celular, una tasa de apoptosis más baja y una expresión de ARNm y proteínas más altas de nefrina y podocina (p<0,05). HKEP puede atenuar el daño de los podocitos inducido por HG, que puede ser uno de los mecanismos de HKEP para atenuar la DN.

Plant Extracts/administration & dosage , Diabetic Nephropathies/drug therapy , Podocytes/drug effects , Powders , Plant Extracts/genetics , Cell Cycle , Blotting, Western , Apoptosis/drug effects , Cell Culture Techniques , Reverse Transcriptase Polymerase Chain Reaction , Glucose
Coluna/Columna ; 20(2): 101-104, Apr.-June 2021. graf
Article in English | LILACS | ID: biblio-1249655


ABSTRACT Approximately 80% of the world population experiences some type of back pain at some point in their life, and in 10% of this population the pain causes chronic disability resulting in a high cost for the treatment of these patients, in addition to compromising their work and social interaction abilities. Current treatment strategies include the surgical procedure for degenerated intervertebral disc resection, the nerve root block and physiotherapy. However, such treatments only relieve symptoms and do not prevent the degeneration of intervertebral discs. Therefore, new therapeutic strategies have emerged and include manipulating cells to recover the degenerated disc. This article will discuss the possible cell therapy alternatives used in the disc regeneration process, featuring a descriptive study of translational medicine that involves clinical aspects of new treatment alternatives and knowledge of basic research areas, such as cellular and molecular biology. Level of evidence V; Expert Opinion.

RESUMO Aproximadamente 80% da população mundial sofre algum tipo de dor nas costas em alguma fase de vida, sendo que em 10% dessa população, as dores acarretam incapacidade crônica, deflagrando alto custo de tratamento desses pacientes, além de comprometer as habilidades de trabalho e convívio social desses indivíduos. As estratégias de tratamento atuais incluem o procedimento cirúrgico por ressecção do disco intervertebral degenerado, bloqueio de raízes nervosas e fisioterapia. Entretanto, tais tratamentos apenas aliviam os sintomas e não impedem que ocorra a degeneração de discos intervertebrais. Portanto, novas estratégias terapêuticas têm surgido e incluem a manipulação de células com o objetivo de recuperar o disco degenerado. No presente artigo, serão discutidas as diferentes possibilidades alternativas de terapias celulares no processo de regeneração discal, caracterizando um estudo descritivo da medicina translacional que envolve aspectos clínicos de novas alternativas de tratamento e o conhecimento de áreas básicas de pesquisa como biologia celular e molecular. Nível de evidência V; Opinião do Especialista.

RESUMEN Aproximadamente 80% de la población mundial sufre algún tipo de dolor de espalda en alguna etapa de la vida, y en 10% de esa población, los dolores causan incapacidad crónica, deflagrando alto costo de tratamiento de esos pacientes, además de comprometer las habilidades laborales y convivencia social de esos individuos. Las estrategias de tratamiento actuales incluyen el procedimiento quirúrgico para la resección del disco intervertebral degenerado, bloqueo de las raíces nerviosas y fisioterapia. Entretanto, tales tratamientos solo alivian los síntomas y no impiden que ocurra la degeneración de discos intervertebrales. Por lo tanto, han surgido nuevas estrategias terapéuticas e incluyen la manipulación de células con el objetivo de recuperar el disco degenerado. En el presente artículo se discutirán las diferentes posibilidades alternativas de las terapias celulares en el proceso de regeneración discal, caracterizando un estudio descriptivo de la medicina traslacional que involucra aspectos clínicos de nuevas alternativas de tratamiento y conocimiento de áreas básicas de investigación como biología celular y molecular. Nivel de evidencia V; Opinión del especialista.

Humans , Cell Culture Techniques , Cell- and Tissue-Based Therapy , Intervertebral Disc
Electron. j. biotechnol ; 50: 59-67, Mar. 2021. ilus, graf, tab
Article in English | LILACS | ID: biblio-1292412


BACKGROUND: Cross talk of tumor­immune cells at the gene expression level has been an area of intense research. However, it is largely unknown at the alternative splicing level which has been found to play important roles in the tumor­immune microenvironment. RESULTS: Here, we re-exploited one transcriptomic dataset to gain insight into tumor­immune interactions from the point of AS level. Our results showed that the AS profiles of triple-negative breast cancer cells co-cultured with activated T cells were significantly changed but not Estrogen receptor positive cells. We further suggested that the alteration in AS profiles in triple-negative breast cancer cells was largely caused by activated T cells rather than paracrine factors from activated T cells. Biological pathway analyses showed that translation initiation and tRNA aminoacylation pathways were most disturbed with T cell treatment. We also established an approach largely based on the AS factor­AS events associations and identified LSM7, an alternative splicing factor, may be responsible for the major altered events. CONCLUSIONS: Our study reveals the notable differences of response to T cells among breast cancer types which may facilitate the development or improvement of tumor immunotherapy.

T-Lymphocytes , Triple Negative Breast Neoplasms , Peptide Chain Initiation, Translational , Gene Expression , Alternative Splicing , Cell Culture Techniques , Receptor Cross-Talk , Transfer RNA Aminoacylation , Transcriptome , Immunotherapy
Braz. j. biol ; 81(1): 37-43, Feb. 2021. graf
Article in English | LILACS | ID: biblio-1153307


Abstract Contamination of primary and cell cultures by mycoplasmas is one of the main economic and biological pitfalls in basic research, diagnosis and manufacture of biotechnological products. It is a common issue which may be difficult to conduct surveillance on. Mycoplasma presence may affect several physiological parameters of the cell, besides being considered an important source of inaccurate and/or non-reproducible scientific results. Each cell type presents characteristical symptoms, mainly morphological, that indicate a contamination by mycoplasma. HEp-2 cells originate from carcinoma of the larynx and are, therefore, part of the respiratory tract, which is one of mycoplasma habitats. Despite the importance these cells in several biological research (evaluation of cell proliferation and migration, apoptosis, antiviral and antitumor compounds), the alterations induced by mycoplasma contamination in HEp-2 cells have not yet been described. Here, we describe the progressive morphological alterations in culture of HEp-2 cells infected with mycoplasma, as well as the-diagnosis of the infection and its treatment. Mycoplasma contamination described within this work led to cytoplasm elongation, cell-to-cell spacing, thin plasma membrane projections, cytoplasmic vacuoles, fusion with neighboring cells, and, finally, cell death. Contamination was detected by fluorescence imaging (DAPI) and PCR reactions. The cultures were treated with BM-Cyclin antibiotic to eliminate contamination. The data presented here will be of relevance to researchers whose investigations involve cell culture, especially respiratory and HEp-2 cells.

Resumo A contaminação de culturas primárias e celulares por micoplasmas é uma das principais armadilhas econômicas e biológicas da pesquisa básica, diagnóstico e fabricação de produtos biotecnológicos. Trata-se de uma contaminação rotineira, mas de difícil acompanhamento. A presença de micoplasma pode afetar vários parâmetros fisiológicos da célula, além de ser considerada uma importante fonte de resultados científicos imprecisos e/ou não reprodutíveis. Cada tipo de célula apresenta sintomas característicos, principalmente morfológicos, que indicam uma contaminação por micoplasma. As células HEp-2 são originárias do carcinoma da laringe e, portanto, fazem parte do trato respiratório, um dos habitats do micoplasma. Apesar da importância destas células em diversas pesquisas biológicas (avaliação da proliferação e migração celular, apoptose, compostos antivirais e antitumorais), as alterações decorrentes da contaminação por micoplasma nestas células ainda não foi descrita. Aqui, descrevemos as alterações morfológicas progressivas na cultura de células HEp-2 infectadas por micoplasma, bem como o diagnóstico da infecção e seu tratamento. A contaminação por micoplasma descrita neste trabalho resultou em alongamento citoplasmático, espaçamento entre células, projeções delgadas da membrana plasmática, vacúolos citoplasmáticos, fusão de células vizinhas e, finalmente, morte celular. A contaminação foi detectada por imagens de fluorescência (DAPI) e reações de PCR. As culturas foram tratadas com antibiótico BM-Cyclin para eliminar a contaminação. Os dados aqui apresentados serão de relevância para pesquisadores cujas investigações envolvem cultura celular, principalmente células respiratórias e HEp-2.

Mycoplasma/genetics , Polymerase Chain Reaction , Cell Culture Techniques , Anti-Bacterial Agents
Braz. j. infect. dis ; 25(5): 101632, 2021. tab, graf
Article in English | LILACS | ID: biblio-1350317


ABSTRACT Emerging human coronaviruses, including the recently identified SARS-CoV-2, are relevant respiratory pathogens due to their potential to cause epidemics with high case fatality rates, although endemic coronaviruses are also important for immunocompromised patients. Long-term coronavirus infections had been described mainly in experimental models, but it is currently evident that SARS-CoV-2 genomic-RNA can persist for many weeks in the respiratory tract of some individuals clinically recovered from coronavirus infectious disease-19 (COVID-19), despite a lack of isolation of infectious virus. It is still not clear whether persistence of such viral RNA may be pathogenic for the host and related to long-term sequelae. In this review, we summarize evidence of SARS-CoV-2 RNA persistence in respiratory samples besides results obtained from cell culture and histopathology describing long-term coronavirus infection. We also comment on potential mechanisms of coronavirus persistence and relevance for pathogenesis.

Humans , RNA, Viral/genetics , COVID-19 , Respiratory System , Cell Culture Techniques , SARS-CoV-2